This project seeks to delineate the fundamental biochemical abnormalities responsible for the accumulation of cystine in various tissues of children with the fatal inherited disease, cystinosis. With our development of a method of prenatal diagnosis the availability of fetal tissues for subculture will enable exploration of a new model system where the defect may be more apparent. Cultured cells from these tissues and cultured skin fibroblasts will obviate the obstacles to biochemical investigation encountered in heterogeneous cell populations. Continuous culture of both of the above model systems together with prolonged fetal organ culture provide means for application of isotopic and enzymologic techniques as well as ultra-microscopic analyses to clarify why cystine accumulates in cells and how this accumulation results in cellular damage. In view of our finding that cultured fibroblasts from normal and cystinotic subjects handle cystine differently, a comprehensive study will be undertaken to delineate the multi-enzyme complex involved in cystine reduction. The control mechanisms governing cystine reduction present in normal cells but apparently absent or defective in cells from cystinotics will be investigated and the role of reduced glutathione in maintaining the cellular homeostasis of cystine will be elucidated. The influx and efflux rates of 35S labeled methionine and ozidized glutathione as well as the intracellular metabolic conversions by cells from normals and cystinotics will be studied in order to delineate the role of such sulfur containing compounds. Radioautographic, electron microscopic, and electron probe analysis with whole cells and tissues and isolated organelles will be utilized to determine intracellular localization of stored cystine and the etiology of structural pathology. The delineation of the biochemical defect in this disease will provide judicious insight into means of prevention and therapy.